1. Field of the Invention
The present invention is in the field of adjuvants and immunostimulating agents. More particularly, the invention pertains to novel triterpene saponin derivatives and their use as adjuvants in vaccine compositions.
2. Related Art
Saponins are glycosidic compounds that are produced as secondary metabolites. They are widely distributed among higher plants and in some marine invertebrates of the phylum Echinodermata (ApSimon et al., Stud. Org. Chem. 17:273-286 (1984)). Because of their antimicrobial activity, plant saponins are effective chemical defenses against microorganisms, particularly fungi (Price et al., CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987)). Saponins are responsible for the toxic properties of many marine invertebrates (ApSimon et al., Stud. Org. Chem. 17:273-286 (1984)). The chemical structure of saponins imparts a wide range of pharmacological and biological activities, including some potent and efficacious immunological activity. In addition, members of this family of compounds have foaming properties (an identifying characteristic), surfactant properties (which are responsible for their hemolytic activity), cholesterol-binding, fungitoxic, molluscicidal, contraceptive, growth-retarding, expectorant, antiinflammatory, analgesic, antiviral, cardiovascular, enzyme-inhibitory, and antitumor activities (Hostettmann, K., et al., Methods Plant Biochem. 7:435-471(1991); Lacaille-Dubois, M. A. & Wagner, H., Phytomedicine 2:363-386 (1996); Price, K. R., et al., CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987)).
Structurally, saponins consist of any aglycone (sapogenin) attached to one or more sugar chains. In some cases saponins may be acylated with organic acids such as acetic, malonic, angelic and others (Massiot, G. & Lavaud, C., Stud. Nat. Prod. Chem. 15:187-224(1995)) as part of their structure. These complex structures have molecular weights ranging from 600 to more than 2,000 daltons. The asymmetric distribution of their hydrophobic (aglycone) and hydrophilic (sugar) moieties confers an amphipathic character to these compounds which is largely responsible for their detergent-like properties. Consequently, saponins can interact with the cholesterol component of animal cell membranes to form pores that may lead to membrane destruction and cell death, such as the hemolysis of blood cells.
Saponin adjuvants from the bark of the Quillaja saponaria Molina tree (quillaja saponins) are chemically and immunologically well-characterized products (Dalsgaard, K. Arch. Gesamte Virusforsch. 44:243 (1974); Dalsgaard, K., Acta Vet. Scand. 19 (Suppl. 69):1 (1978); Higuchi, R. et al., Phytochemistry 26:229 (1987); ibid. 26:2357 (1987); ibid. 27:1168 (1988); Kensil, C. et al., J. Immunol. 146:431 (1991); Kensil et al., U.S. Pat. No.5,057,540 (1991); Kensil et al., Vaccines 92:35 (1992); Bomford, R. et al., Vaccine 10:572 (1992); and Kensil, C. et al., U.S. Pat. No. 5,273,965 (1993)). From an aqueous extract of the bark of the South American tree, with Quillaja saponaria Molina, twenty-two peaks having saponin activity were separated by chromatographic techniques. The predominant purified saponins were identified as QS-7, QS-17, QS-18 and QS-21. QS-21 was later resolved into two additional peaks, each comprising a discrete compound, QA-21-V1 and QA-21-V2. See Kensil et al., U.S. Pat. No. 5,583,112 (1996).
These saponin adjuvants are a family of closely related O-acylated triterpene glycoside structures. They have an aglycone triterpene (quillaic acid), with branched sugar chains attached to positions 3 and 28, and an aldehyde group in position 4. Quillaja saponins have an unusual fatty acid substituent (3,5-dihydroxy-6-methyloctanoic acid) as a diester on the fucose residue of the C-28 carbohydrate chain. This ester is hydrolyzed under mildly alkaline conditions or even at physiological pH over short periods of time to produce deacylated saponins including DS-1 and DS-2 (Higuchi et al., Phytochemistry 26:229 (1987)); (Kensil et al., Vaccines 92:35-40 (1992)). More severe hydrolysis of these saponins using strong alkalinity (Higuchi et al., Phytochemistry 26:229 (1987)) or prolonged hydrolysis (Pillion, D. J., et al., J. Pharm. Sci., 85:518-524 (1996)) produces QH-957, the result of hydrolysis of the C-28 ester. The triterpenoid hydrolysis by-products have hydrophobic/hydrophilic properties differing from those of QS-21; these differences result in altered micellar and surfactant properties.
The loss of the fatty acid ester on fucose is of particular interest since it greatly reduces the adjuvant properties of QS-21 and other related quillaja saponins. A comparison of the humoral response elicited by quillaja saponins and its deacylated by-product shows that, although quillaja saponins stimulates a strong primary Th1 antibody response, their deacylated by-products elicit only a poor primary immune response (Marciani et al., unpublished observations). This poor primary response is similar to that produced by gypsophila and saponaria saponins that are naturally non-acylated (Bomford, R., et al., Vaccine, 10:572-577 (1992)). Subsequent immunizations with deacylated quillaja saponins do produce good secondary Th1 antibody response (Marciani et al., unpublished observations) that is similar to that produced by gypsophila or saponaria saponins (Bomford, R., et al., Vaccine, 10:572-577 (1992)). However, immunizations with deacylated QS-21 or quillaja saponins fail to stimulate either the production of cytotoxic T lymphocytes (CTLs) (Pillion et al., 1995), or the priming of T lymphocytes (Marciani et al., unpublished observations). These results show the hydrophobic acyl group on fucose of the quillaja saponins is also an extremely critical structural feature for stimulation of a primary immune response as well as for stimulation of cell-mediated immunity (CMI) (Press, J. B., et al., Studies in Natural Product Chemistry, Atta-Ur-Rahman, ed.: Elsevier, Amsterdam 21:1-50 (1999)). In addition, this acyl group and its ability to hydrolyze is a cause of at least part of the toxicity of quillaja saponins (Press, J. B., et al., Studies in Natural Product Chemistry, Atta-Ur-Rahman, ed.: Elsevier, Amsterdam 21:1-50 (1999)).
The immune system may exhibit both specific and nonspecific immunity (Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston (1997)). Generally, B and T lymphocytes, which display specific receptors on their cell surface for a given antigen, produce specific immunity. The immune system may respond to different antigens in two ways: 1) humoral-mediated immunity, which includes B cell stimulation and production of antibodies or immunoglobulins [other cells are also involved in the generation of an antibody response, e.g. antigen-presenting cells (APCs; including macrophages), and helper T cells (Th1 and Th2)], and 2) cell-mediated immunity, which generally involves T cells including cytotoxic T lymphocytes, although other cells are also involved in the generation of a CTL response (e.g., Th1 and/or Th2 cells and APCs).
Nonspecific immunity encompasses various cells and mechanisms such as phagocytosis (the engulfing of foreign particles or antigens) by macrophages or granulocytes, and natural killer (NK) cell activity, among others. Nonspecific immunity relies on mechanisms less evolutionarily advanced (e.g., phagocytosis, which is an important host defense mechanism) and does not display the acquired nature of specificity and memory, hallmarks of a specific immune response. Nonspecific immunity is more innate to invertebrate systems. In addition, cells involved in nonspecific immunity interact in important ways with B and T cells to produce an immune response. The key differences between specific and nonspecific immunity are based upon B and T cell specificity. These cells predominantly acquire their responsiveness after activation with a specific antigen and have mechanisms to display memory in the event of future exposure to that specific antigen. As a result, vaccination (involving specificity and memory) is an effective protocol to protect against harmful pathogens.
A critical component of inactivated vaccines, including subunit vaccines, is an adjuvant. Immune adjuvants are compounds that, when administered to an individual, increase the immune response to an antigen in a test subject to which the antigen is administered, or enhance certain activities of cells from the immune system. Some antigens are weakly immunogenic when administered alone or are toxic to a subject at concentrations that evoke useful immune responses in a subject. In these cases, an immune adjuvant can be used to enhance the immune response of the subject to the antigen by making the antigen more strongly immunogenic. The adjuvant may also allow the use of a lower dose of antigen to achieve a useful immune response in a subject.
Immune adjuvants can modify or immunomodulate the cytokine network, up-regulating the humoral and cellular immune response. Humoral response elicits antibody formation. Cellular immune response involves the activation of T cell response, Th1 or Th2, to mount this immune response. Th1 responses will elicit complement fixing antibodies and strong delayed-type hypersensitivity reactions associated with IL-2, IL-12, and .gamma.-interferon. Induction of cytotoxic T lymphocytes (CTLs) response also appears to be associated with a Th1 response. Th2 responses are associated with high levels of IgE, and the cytokines IL-4, IL-5, IL-6, and IL-10. The aldehyde-containing saponins such as those from quillaja induce a strong Th1 antibody response. However, some of their analogs may induce a Th2 response.
Adjuvants that have been used to enhance an immune response include aluminum compounds (all generally referred to as "alum"), oil-in-water emulsions (often containing other compounds), complete Freund's adjuvant (CFA, an oil-in-water emulsion containing dried, heat-killed Mycobacterium tuberculosis organisms), and pertussis adjuvant (a saline suspension of killed Bordatella pertussis organisms). These adjuvants generally are thought to have their mechanism of action by causing a depot of antigen and permitting a slow release of the antigen to the immune system, and by producing nonspecific inflammation thought to be responsible for their observed activity (Cox, J. C., et al., Vaccine 15:248-256 (1997)). Some saponins have been shown to have different types of immune stimulating activities, including adjuvant activity. These activities have been reviewed previously (Shibata, S., New Nat. Prod. Plant Pharmacol. Biol. Ther. Act., Proc. Int. Congr. 1st, 177-198 (1977); Price, K. R., et al. CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987); Schopke, Th., & Hiller, K., Pharmazie 45:313-342 (1990); Lacaille-Dubois, M. A., et al., Phytomedicine 2:363-386 (1996)).
U.S. Pat. No. 5,583,122 describes conjugates in which poorly immunogenic proteins are covalently attached to purified, acylated Quillaja saponin fraction via the carboxyl group of 3-O-glucuronic acid. Addition of free quillaja saponins to these conjugates induced a higher immune response.
PCT Published Application No. WO 90/03184 describes an immunostimulating complex (ISCOM) comprising at least one lipid and at least one saponin, and that may optionally include adjuvants in addition to the saponin. These matrices are taught to be useful as immunomodulating agents and vaccines. The lipid and saponin are in a physical association, rather than covalently attached to one another. Quil A (a Quillaja saponin extract) is the preferred saponin. The reference additionally teaches that it is beneficial to add adjuvants (in addition to Quil A) to the ISCOM matrix. The reference teaches that an adjuvant lacking suitable hydrophobic properties may be modified to comprise a hydrophobic domain for incorporation into the ISCOM matrix.
Bomford, R. et al., Vaccine 10:572-577 (1992) teaches that lipids can be mixed with a variety of saponins to form ISCOM's. The reference teaches that Quillaja saponins, Gypsophila saponins and Saponaria saponins were the only saponins tested that were adjuvant active.
There remains a need for adjuvants that have enhanced adjuvanticity and lower toxicity. Thus, it would be of commercial interest to develop adjuvants which are less toxic, chemically more stable, and with equal or better adjuvant properties than existing adjuvants.